Abstract Food allergy is mediated by antigen crosslinking of IgE antibodies bound to mast cells (MC) and is often associated with intestinal dysbiosis. IgE sensitization to food is not by itself sufficient to drive food anaphylaxis. Intestinal MC load, which controls intestinal permeability and systemic antigen absorption, is an important determinant. The guanine nucleotide exchange factor, DOCK8, is important for STAT3 activation, IL-22, and IL- 17 production. As food allergy and anaphylaxis are cardinal features of DOCK8 deficient patients, DOCK8 deficiency offers an attractive disease model for identifying mechanisms underlying food allergy. We find elevated serum tryptase levels in DOCK8 deficient patients. Dock8-/- mice exhibit exaggerated IgE- mediated anaphylaxis to oral, but not i.p. antigen challenge, as well as intestinal MC expansion, which we show is neither a consequence of a cell intrinsic DOCK8 deficiency within MCs, nor dependent on IgE. Mice with selective deficiency in DOCK8 in T cells phenocopy Dock8-/- mice, and both exhibit similar intestinal dysbiosis T cells in the small intestine (SI) lamina propria (LP) of Dock8-/- mice have decreased expression of Il22 and Il17 and increased expression of Il4 and Il13. DOCK8 deficient SI epithelium has decreased expression of antimicrobial peptide (AMP) genes, but increased numbers of tuft cells and elevated Il25 expression. Treatment with either sIL-22 or an oral antibiotic cocktail normalizes intestinal MC expansion, suggesting a critical role for dysbiosis driven by type 17 cytokine deficiency. IL-25 and IL-4 blockade reduce intestinal MC load and passive oral anaphylaxis in Dock8-/- mice, suggesting a critical role for IL-25, which promotes type 2 cytokine production by ILC2s and Th2 cells, and for IL-4, which acts on MCs. Importantly, germ free (GF) WT recipients of fecal microbiota transplantation (FMT) from conventionally raised Dock8-/- mice have significantly greater intestinal MC load compared to GF WT recipients of FMT from conventionally raised WT controls. Thus, our proposed studies investigate the mechanisms by which dysbiosis in DOCK8 deficiency may increase intestinal MC load and thereby promote anaphylaxis are relevant to patients with classic food allergy. We will test the hypotheses that 1) reduced production of type 17 cytokines due to defective STAT3 activation in DOCK8 deficiency results in intestinal dysbiosis, 2) dysbiosis causes intestinal tuft cells to produce IL-25, which drives type 2 cytokine production by ILC2s and Th2 cells, and 3) IL-4, in turn, drives intestinal MC expansion and activation, thereby promoting anaphylaxis to ingested antigen. We will make use of transgenic mice and take advantage of our access to DOCK8 deficient patients and previously well-characterized fecal samples from children with classic food allergy to test our hypotheses. The proposed studies will delineate how immune mechanisms important for maintaining the intestinal microbiome regulate intestinal MC expansion and activation, and thereby the severity of food allergy, thus identifying novel strategies for the prevention and treatment of food allergy.